Many biomarkers are responsive to oxidative stress and modulating effects of antioxidant dietary supplements, but the disease relevance of these markers is uncertain because of the lack of known functional or mechanistic linkages. We propose that mutation is a disease-relevant target endpoint because of its known association with aging and causal relationship to cancer. We also propose that mitochondrial DNA (mtDNA) mutation leading to heteroplasmy is likely to be an ultra sensitive sentinel biomarker of oxidative stress. ("Heteroplasmy" is when a new mtDNA mutation arises leading to a mixed intracellular mtDNA population.) MtDNA is unusually sensitive to oxidative damage because of proximity to the main source of cellular oxidants. We (C. Calloway) developed a sensitive assay of mtDNA mutation/heteroplasmy which is widely used in forensics. Here, she proposes to use a novel, ultra sensitive sequencing method to characterize the full spectrum of mutations in mtDNA. We propose to evaluate the sensitivity of this assay to oxidative stress, the specificity of mutational spectra resulting from different causes of oxidative stress, and the responsiveness of the assay to modulatory effects of antioxidants. Three biomarker assays will be evaluated in F344 rats: oxidant-induced mtDNA damage (with B. Van Houten);metabolomics by LCMS (J. Suh) for changes to redox status;and malondialdehyde (MDA) by GCMS for oxidative stress developed in our lab and widely used. The time course and sensitivity of these assays will be compared following physiologically relevant oxidative stresses induced by old age and by iron deficiency, which we expect to be different quantitatively and qualitatively. These effects will be compared to CCl4 stress, which was the oxidative stress utilized in the NIHinitiated multi-center BOSS study using the common oxidative stress assays, including our GCMS-MDA assay. MtDNA mutations resulting from oxidative stress should be detected because of the exquisite sensitivity of the mtDNA mutation/heteroplasmy assay, as will different mutational spectra resulting from different stressors. Reversal of heteroplasmy, due to turnover of dysfunctional mitochondria, will be examined on removal of oxidative stress. Understanding the time course and relative magnitude of these effects on mutation/heteroplasmy as compared to the effects of antioxidant supplementation on other biomarkers (e.g., mtDNA damage, re-establishment of a normal metabolomic redox profile) will be a key aspect of the project. If mtDNA mutation/heteroplasmy is a sensitive biomarker of oxidative stress and is responsive to dietary antioxidant supplementation, it will be a functional biomarker linked to disease and it will serve as a reference against which other more generic oxidative stress biomarkers can be calibrated. It will also be useful as a sentinel for oxidative mutations of genomic DNA, which are expected to be in much smaller numbers and would not be reversible by antioxidant supplementation. The mtDNA mutation/heteroplasmy assay is suitable for use in clinical trials using human hair, buccal swabs, or small amounts of blood. PUBLIC HEALTH RELEVANCE: Oxidative stress causes mitochondrial dysfunction which is associated with many degenerative diseases of aging and with the aging process itself. The mitochondrial DNA mutation/heteroplasmy assay being proposed is a novel, highly sensitive method to detect functional cellular changes caused by oxidative stress. The assay will help scientists better understand how oxidative stress leads to disease and will also provide a way to measure functional benefits of dietary antioxidant supplementation.